The last decade has seen great progress in therapeutic and prophylactic approaches based on vaccination against antigens present on tumor cells and infectious pathogens. Despite the advances made in the identification of new antigens and the elucidation of mechanisms that allow for targeted immune responses against such antigens, a number of challenges remain to be resolved. One obstacle, in particular, is the generation of sufficiently potent immune responses even after the identification of new antigens, as many promising antigenic targets have been shown to be only weakly immunogenic.
To elicit an immune response, antigens have to be taken up and processed by a special type of cell, the so-called antigen-presenting cells (APCs). The three cell types able to present antigens are dendritic cells, macrophages, and B-lymphocytes. The uptake is facilitated by phago- or endocytosis and the processing is done in vesicles or the cytosol. Key players in the presentation of the antigen on the cell surface are a class of proteins termed major histocompatibility complex proteins (MHC). These molecules are encoded by the most polymorphous gene family in the human genome located on chromosome 6 and can be subdivided into class I and II. The topology of both classes of molecules is such that they can bind and present as broad a spectrum of peptides 8 to 16 amino acids in length as possible. Thus, a very effective immunosurveillance is ensured. The copy number of a defined antigenic molecule on the surface of an antigen-presenting cell is quite low (about one hundred), given the total number of about ten thousand receptors, but this feature accounts for a very heterogeneous mixture of antigenic peptides on the surface of each APC.
MHC class I molecules bind and present samples of peptides, including endogenous as well as translated and processed viral or tumor antigens and activate the cellular, cytotoxic immune response via CD8+ (cytotoxic) T-cells. Autoimmunity against endogenous molecules is normally prevented by the negative selection process of the immune cells in the thymus, the bone marrow, and the lymphatic system. MHC class II molecules bind and present peptides, which are ingested from the immediate cellular environment and processed by a variety of enzymes in vesicles, generated by the fusion of lysosomes and phagosomes. Peptides bound to class II molecules activate CD4+ (helper) T-cells, which in turn activate B-cells, thus inducing the humoral immune response, and/or CD8+ T-cells, thus inducing a cellular immune response.
A peptide has the ability to provoke a specific cellular immune response if it is capable of binding to MHC molecules and has the ability to be recognized by CD8+ or CD4+ T-cells or it has the ability to induce a humoral response if it is recognized by a B-cell via membrane bound immunoglobulin. Immunogenicity of an antigen is defined by many variables including size, structure, stability, difference to endogenous molecules, adjuvant presence and the immune condition of the organism as well as other genetic factors. When the antigen is no foreign protein or peptide, it is normally not recognized by T-cells, since these cells are not present or are selected not to react with endogenous proteins, and its presentation in the MHC complex on the surface of an antigen-presenting cell is not sufficient to provoke an immune response.
The use of proteins or glycoproteins in the development of new and effective vaccines is controversial as, in several cases, it has proven to be either ineffective or hazardous to the inoculated organism. This property is due to a lack of immunogenicity based on a non-favorable size, structure, stability, or homology to endogenous proteins, or to the inclusion of non-protective epitopes. In addition, native peptides have a low systemic stability and are rapidly degraded by proteases. Another disadvantage is that it is not guaranteed that the peptide is capable to bind to major histocompatibility complex (MHC) proteins class I or II, which is absolutely necessary for the elicitation of an immune response.
Because such weak immune responses offer little clinical benefit, the development of effective immune response potentiating compounds to enhance the immunogenicity of target antigens has become a goal of increasing therapeutic and prophylactic importance.
Immune response potentiating compounds are classified as either adjuvants or cytokines. Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes. Adjuvants of many kinds are well known in the art; specific examples include Freund's (complete and incomplete), components of the cells wall of mycobacterias (Bacillus calmette-guerin, Mycobacterium vaccae, corynebacterium parvum), lipopolysaccharides such as Lipid A, monophosphoryl Lipid A, LPS or LPS derivates or MF59 (Chiron), and various oil/water emulsions (e.g. IDEC-AF). Other currently used adjuvants include: mineral salts or mineral gels such as aluminium hydroxide, aluminium phosphate, and calcium phosphate, surface active substances such as lysolecithin, pluronic polyols, polyanions, keyhole limpet hemocyanins, and dinitrophenol, immunostimulatory molecules, such as saponins (QS-21 (SmithKline Beecham) ISCOMs), muramyl dipeptides and tripeptide derivatives, CpG dinucleotides, CpG oligonucleotides, lipopeptides/lipoproteins, cholera toxin and polyphosphazenes, particulate and microparticulate adjuvants, such as emulsions, cochleates, or immune stimulating complex mucosal adjuvants.
Cytokines are also useful in vaccination protocols as a result of lymphocyte stimulatory properties. Many cytokines useful for such purposes will be known to one of ordinary skill in the art, including interleukin-2 (IL-2), IL-12, GM-CSF, and many others.
Immunization strategies to efficiently prime CD8+ T cell responses have come into focus of research activity in current vaccinology. Recent advances in the development of potent adjuvants resulted in two main, alternative approaches, i.e. peptide- and DNA-based vaccine formulation. However, despite their promise as powerful adjuvants, the administration of both can exert toxic effects on the host, which limits their usefulness in the clinical context. It would thus be desirable to develop adjuvant systems that provide potent immune stimulation while reducing or eliminating the risk of toxic side effects.
WO 98/50071 discloses adjuvant properties of virus-like particles (VLPs), which enhance the humoral and/or cell-mediated immune response in vertebrates if administered with a selected antigen. VLPs are non-replicating empty viral shells composed of one or more capsid, coat, shell, surface and/or envelope proteins. According to the disclosure it was found that the selected antigen does not have to be entrapped in the VLP for the VLP to exert its adjuvant or coadjuvant effect.
European Patent No. 1550458 describes a method to enhance the specific immune response against an antigen, which is administered in a liposome together with a first conventional adjuvant, either also comprised in the first liposome or in a second liposome, by the coadministration of a second conventional adjuvant in free or liposomal form. The inventors found out that surprisingly the adjuvant action observed for two free adjuvants could be further enhanced if at least one of the adjuvants and the antigen was comprised in the same or separate liposomes.
WO 2004/045582 discloses a fusogenic vesicle containing encapsulated antigen. The fusogenic vesicle is the product of fusing liposomes with virosomes bearing hemagglutinin (“HA”) fusion proteins from different virus strains. The fusion of liposomes and virosomes is triggered by conditions of low pH (about pH 4.5-5).
WO 2006/085983 discloses virus replicating particles (“VRPs”) which can act as an adjuvant to enhance an immune response against an immunogen not presented or expressed by the virus. The inventors state that the adjuvant effect is dependent on the VRP's ability to replicate, meaning that the VRPs disclosed contain either unmodified or modified portions of a viral genome.
Schumacher et al. (Vaccine 22:714-723, 2004) found that Immunopotentiating reconstituted influenza virosomes (IRIV), the adjuvant capacity of which in the induction of humoral immune responses has been demonstrated before, are also capable to enhance the T cell-mediated immune response. More specifically, Schumacher and colleagues could show IRIV adjuvant activity in the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) in vitro. This capacity was found to mainly rely on the stimulation of CD4+ T cell reactivity specific for viral proteins.
The adjuvant properties of IRIVs are well known in the art, for example from WO 92/19267, wherein an adjuvant effect of the IRIVs for an antigen coupled thereto is disclosed.
However, although the use of virosomes as adjuvants has a number of advantages, for example low toxicity and high immunogenicity, one of the problems in current virosome technology is the lack of methods for the efficient entrapment of a solute, e.g. protein, nucleic acid, or pharmaceutical drug. At the lipid concentration at which virosomes are produced (˜1 mM lipid), and given their mean diameter of approximately 200 nm, less than 1% of the aqueous phase will be entrapped within the virosomes (Schoen et al., J. Liposome Res. 3: 767-792, 1993). Such low entrapment rates reduce virosome-mediated efficiency of antigen, drug, or gene delivery to cells. Thus, one of the problems in current virosome technology is the lack of methods for the efficient entrapment of a solute, e.g. protein, nucleic acid, or pharmaceutical drug.
Therefore, there exists still a need for efficient and cost-effective adjuvant systems to enhance the immunogenicity of otherwise weakly immunogenic antigenic molecules, preferably combined with safe and efficient delivery characteristics.